Immune responses must be regulated to ensure effective pathogen elimination and self tolerance. ICOS and PD-L1 play key roles in regulating this critical balance between T cell activation and tolerance. During the current funding period, we have been investigating how ICOS and PD-L1 in regulate pathogenic vs. protective T cell responses. Our studies point to novel roles for both ICOS and PD-L1 In sustaining the function of regulatory T cell cells and maintaining T cell tolerance. We have found that ICOS does not control the induction of regulatory CD4 T cells during mucosal tolerance, but instead, appears necessary for sustaining CD4 regulatory cells during mucosal tolerance. ICOS, c-Maf and IL-21 coordinately act to promote differentiation of IL-10 producing regulatory type 1 (Tr1) cells, but ICOS appears to be crucial for maintaining IL-27 driven, IL-10 producing Tri cells. These findings lead us to hypothesize that ICOS, c-maf and lL-27 wori< in concert to regulate mucosal tolerance. Likewise, we have identified mechanisms by which PD-L1 controls tolerance: PD-L1 limits activation of self-reactive T cells, function of self-reactive effector cells and promotes induction and maintenance of adaptive regulatory T cells. PD-L1 on non-hematopoietic cells promotes tissue tolerance. PD-L1 and PD-1 have become new therapeutic targets in cancer and chronic infection, since blockade of PD-1 or PD-L1 can activate anti-viral or anti tumor immunity. In view of their key roles In regulating tolerance, further studies are needed to determine how to effectively minimize the risk of immune-mediated tissue damage and autoimmunity, while modulating PD-L1 and PD-1 to enhance virus or tumor control. Our discovery of the PD-L1 :B7-1 pathway leads us to ask whether PD-L1 :PD-1 and PD- L1:B7-1 interactions have unique or overiapping roles in T cell tolerance, and investigate the function of PD- L1 on T cells during tolerance. We hypothesize that PD-L1:B7-1, as well as PD-L1:PD-1 interactions, inhibit self-reactive T cell responses, and that PD-L1 has a T cell intrinsic role in controlling T cell responses. To test these hypotheses, our Specific Aims are to: 1) Investigate the inter-relationships among ICOS, IL-27 and c-maf during mucosal tolerance; 2) Analyze the functional significance of the novel PD-L1:B7-1 pathway and relative contributions of PD-L1:B7-1 and PD-L1 interactions in regulating T cell tolerance and autoimmunity; 3) Investigate the role of PD-L 1 onT cells in regulating T cell tolerance; and 4) Dissect the roles of PD-L 1 on specific cell types in mucosal tolerance. These studies should complement each other to further our understanding of mechanisms that control tolerance and autoimmunity, and provide therapeutic insights.